Abstract: Verticillin A is a natural product isolated from fungal cultures and has displayed potent antibiotic, antiviral, nematocidal, and anticancer properties in vitro. While in vivo studies have been limited due to sparse supply, the in vivo efficacy data that does exist demonstrates potent anti-tumor activity in murine cancer models. The current study aims to investigate the pharmacokinetics and bioavailability of verticillin A in mice to provide guidance for further efficacy assessment in mouse models. A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of verticillin A in mouse plasma. Sample preparation was accomplished through protein precipitation, and chromatographic separation was achieved on an Agilent Zorbax Extend C18 column with a security guard cartridge C8 using a binary gradient with mobile phase A (water/0.1% formic acid) and B (ACN/0.1% formic acid) at a flow rate of 400 µl/min. Elution of verticillin A and internal standard, hesperetin, occurred at 4.87 and 2.06 min, respectively. The total chromatographic run time was 8 min, and the assay was linear in the concentration range of 1–1000 nM. The within- and between day precisions and accuracy were in the range of 2.58–8.71 and 90–105%, respectively. The assay was applied to determine plasma drug concentration in a mouse pharmacokinetic study. It was found that intraperitoneal dosing of 3 mg/kg resulted in high systemic exposure and achieved Cmax of 110 nM with plasma concentrations sustained above 10 nM for the 24-h duration of the study. Intravenous and oral dosing achieved observed Cmax of 73 nM and 9 nM, respectively. Oral dosing resulted in an approximate 9% bioavailability. Comparing with previously published in vitro studies that demonstrated verticillin A is active in the 20 nM to 130 nM range, the pharmacokinetic data demonstrate similar levels are achieved in mouse plasma via intravenous or intraperitoneal dosing routes.